1 INTRODUCTION
1.4 RELEASE OF STERIODS THROUGH THE PLASMA MEMBRANE
The steroid biosynthesis, the regulation of steroid release and their physiological effects on the function in the body have been topics of intensive research since the first description of adrenals by Bartholomeo Eustachius in Tabulae Anatomicae, which was later edited and published (Eustachius 1774). In the recent years, there have been numerous additions to the knowledge of different biosynthetic pathways and their action on their target organs and cells. In contrast, the release of steroids from the steroid synthesizing cells into the blood and their entry into the target cells is poorly understood. It has long been assumed that the release occurs via simple diffusion or exocytosis, based on the lipophilic structure of steroid hormones. The idea of exocytosis or any relevant storage of cortisol has never been supported by direct morphological evidence (Bassett et al. 1980; Gemmell et al. 1977). However, in vitro studies have demonstrated retention of steroids against a concentration gradient at the plasma membrane (Inaba et al. 1974; Whitehouse et al. 1971).
The transporter-mediated uptake of glucocorticoids was first demonstrated by Rao (Rao et al. 1976). The uptake of cortisol into isolated liver cells was temperature dependent, showed saturation kinetics, was inhibited by cortisone and corticosterone, and was significantly decreased by metabolic inhibitors and sulfhydryl reagents. The uptake was independent of sodium and showed no effect by ouabain.
The uptake into the liver cells from the external media was a rapid process, and showed protein mediated characteristics. As the specific glucocorticoids binding proteins are localised in the cytoplasm, their involvement in uptake of cortisol was not suggested. In conclusion the transport of cortisol into the liver cells seemed to be in part a carrier-mediated action (Rao et al. 1976). Using the stop-flow peritubular capillary microperfusion method, Ullrich and co-workers (Ullrich et al. 1991) described the inhibition of contraluminal transport of radiolabeled p-aminohippurate (PAH) in the proximal tubule of the rat kidney by cortisol. In these experiments the basolateral uptake of PAH and of labeled cortisol into proximal tubule cells was inhibited by probenecid. These studies indicated the involvement of transporter proteins in the translocation of glucocorticoids through the plasma membrane.
Recent studies showed the inhibition of cortisol release from primary cultures of bovine adrenocortical cells by probenecid. Further investigations on bovine adrenocortical cells demonstrated an uptake of radioactively labeled PAH, which was inhibited by probenecid. The uptake of organic anions (i.e. PAH) into the cells as well as the cortisol release from the cells was stimulated by ACTH (Steffgen et al. 1999).
These data indicated the possible involvement of organic anion transporters in cortisol release. Investigations on the molecular level of rat adrenals revealed the expression of organic anion transporter 1 (OAT1). In-situ hybridizations and immunohistochemical analyses localized rat OAT1 to the zona fasciculata of the adrenal cortex, where cortisol synthesis and release take place. Importantly, OAT1 mRNA expression was strongly increased by treatment of rats with ACTH in vivo (Beery et al. 2003). All these evidences emphasize the possible involvement of a transport mechanism in steroid hormone release from adrenocortical cells.
1.4.1 Organic anion transporter (OAT) family
One of the possible candidates for glucocorticoid export from adrenocortical cells is the organic anion transporter. Organic anion transporters (OAT) perform an important task in the renal secretion of a wide range of organic anions, such as endogenous metabolic waste products and exogenous potentially toxic compounds, especially drugs such as loop diuretics, non-steroidal anti-inflammatory drugs, and ß-lactam antibiotics. The secretion of these permanently negatively charged organic anions occurs in the renal proximal tubule. These transporters are conserved through evolution from Caenorhabditis elegans to mammals (Burckhardt et al. 2003).
The topological organization of OAT1 proteins within the membrane is unknown, however secondary structure studies predict them to span the membrane twelve times and to have two large hydrophilic loops between trans-membrane domains one and two and between trans-membrane domains six and seven. Both C and N-termini located in the cytosol (Burckhardt et al. 2000b).
Transport of organic anions through OAT1 and OAT3 into the proximal tubule cells at the basolateral membrane is a tertiary active exchange against α-ketoglutarate. The intracellular α-ketoglutarate level is maintained by metabolism and by transport into the cells across the basolateral as well as the luminal membrane, mediated by sodium-dicarboxylate co-transporters. The inwardly directed gradient for sodium is maintained by the primary active, basolaterally located Na+/K+ATPase (Burckhardt et al. 2000a; Burckhardt et al. 2001b).
Meanwhile more than four isoforms of the OAT family (OAT1, OAT2, OAT3 and OAT4) are known in their molecular structure and are functionally characterized.
Since 1997 several groups cloned OAT1 orthologs from different species like the rat, Winter flounder, human, rabbit and pig (Bahn et al. 2002a; Hagos et al. 2002;
Sekine et al. 1997; Wolff et al. 1997). The human OAT1 ortholog was cloned in 1998/99 (Hosoyamada et al. 1999; Reid et al. 1998) and functionally characterized.
The model substrate of OAT1 is PAH which exhibits a high affinity with Km values in
the range of 4 to 20 µM. An interaction of human OAT1 with cortisol has not yet been tested directly.
Figure 1.2 Secretion of organic anions in a model renal proximal tubule cell.
The organic anions (OA–) are taken up from the interstitium/blood into proximal tubule cells. The secretory pathway for organic anions through OAT1 and OAT3 exchange an extracellular OA– against an intracellular α-ketoglutarate (α-KG2–). The α-ketoglutarate released by OAT1 and OAT3 is pumped back into the cell by NaDC-3, and the three Na+ ions co-transported with α-ketoglutarate are removed by the Na+,K+-ATPase. Additionally the intracellular pool of α-ketoglutarate is maintained by its generation through metabolism. Several transporters for organic anions have been identified on the luminal membrane such as the urate/anion exchanger (URAT1), multidrug resistance protein 2 (MRP-2), sodium-phosphate transporter (NPT), and OAT4.
Another member of OAT family is OAT2, which was initially cloned as a novel liver transporter (NLT) (Sekine et al. 1998b; Simonson et al. 1994) and transports α-ketoglutarate, prostaglandins, salicylate, and PAH (Burckhardt et al. 2003). As with OAT1, the interaction of OAT2 with glucocorticoids was not tested. Several orthologs of OAT3 were cloned and functionally characterized (Cha et al. 2001; Hasegawa et al. 2002; Race et al. 1999). The transport mechanism of OAT3 is similar to that of OAT1, as it exchanges organic anions against dicarboxylates like glutarate and α-ketoglutarate (Bakhiya et al. 2003; Sweet et al. 2002). Immunohistochemical analyses revealed the localization of hOAT1 and hOAT3 at the basolateral
membrane of proximal tubule cells. Real-time PCR data of human kidney cortex showed a two fold higher expression of hOAT3 compared to hOAT1 and a more than tenfold higher expression than that of hOAT2 and hOAT4 (Motohashi et al. 2002).
The expression analysis of rat kidney showed a higher expression of rOAT1 than of rOAT3, while, the hOAT3 expression is stronger than hOAT1 in human kidney (Bossuyt et al. 1996b). The human OAT3 has a high affinity for estrone sulfate (ES), dehydroepiandosterone sulfate (DHEAS), and 17β-estradiol-17βD-glucuronide, but exhibits low affinity for PAH (Cha et al. 2001; Race et al. 1999; Sugiyama et al.
2001). Corticosterone inhibited estrone sulfate transport by hOAT3 (Cha et al. 2001).
These facts suggest the interaction of OAT3 in transport of sulfated and glucuronidated steroid hormones and possibly in translocation of glucocorticoids into the cells. The fourth member of organic anion transporter family, OAT4, was cloned from a human kidney cDNA library and was evident from placenta too (Cha et al.
2000). Up to now there is no comparable ortholog for hOAT4 from other species, and it seems that OAT4 represents a human-specific member of the organic anion transporter family. Human OAT4 shares substrate specificity with hOAT3. It has a very high affinity for DHEAS and estrone sulfate with Km of 0.6 and 1.0 µM, respectively, but does not show any affinity for glucuronic acid-conjugated steroids (e.g. β-estradiol-3β-D-glucuronide). The estrone sulfate uptake by OAT4 was inhibited by corticosterone (Cha et al. 2000), pointing to a possible interaction of hOAT4 with glucocorticoid translocation.
1.4.2 Organic anion transporter polypeptide (OATP) family
Another possible candidate for the steroid release from human adrenocortical cell are organic anion transporter polypeptides (OATP). OATPs are selectively expressed in rodent and human livers, where they are involved in the hepatic clearance of albumin-bound compounds from portal blood plasma (Meier et al. 2002). OATPs show multiple tissues expression including the blood–brain barrier (BBB), choroid plexus, lung, heart, intestine, kidney, placenta and testis (Tamai et al. 2000). A large number of members of the OATP family have not been characterized in detail on the functional, structural and genomic levels. However, initial studies with individual OATPs indicate that many members of this transporter family represent polyspecific organic anion carriers with partially overlapping substrate preferences for a wide range of amphipathic organic solutes including bile salts, organic dyes, steroid conjugates, thyroid hormones, anionic oligopeptides, numerous drugs and other xenobiotic compounds (Hagenbuch et al. 2003; Ublick et al. 2000; Kullak-Ublick et al. 2001; Meier et al. 1997). The hydropathy analysis showed that all
OATPs have 12 transmembrane (TM) domains, which have yet to be proven by experimental evidence (Jacquemin et al. 1994).
The first human OATP cloned from human liver was OATP-A (Kullak-Ublick et al.
1995). OATP-A protein is expressed at the blood-brain barrier along the border of brain microvessels and capillary endothelial cells. OATP-A mRNA was detected in brain, lung, liver, kidney, and testis (Kullak-Ublick et al. 2001). As compared to other human OATPs, OATP-A exhibits broad substrate specificity and transports bile acids, bromosulfophthalein (BSP), steroid hormone conjugates, thyroid hormones, oligopeptides, ouabain, and amphipathic organic cations. Functional studies with OATP-A showed that it does not transport cortisol (Bossuyt et al. 1996b). Another member of OATP family, OATP-B, was isolated from human brain. The mRNA for OATP-B showed broad tissue distribution: liver, spleen, placenta, lung, kidney, heart, ovary, small intestine, and brain (Kullak-Ublick et al. 2001). The functional characterization showed that OATP-B mediates high affinity uptake of BSP and also transports estrone-3-sulfate and DHEAS, but not bile acids.
OATP-C and OATP-8 were cloned from human liver. OATP-C protein is expressed at the basolateral domain of human hepatocytes. The substrate specificity of OATP-C includes taurocholate, bilirubin, BSP, steroid hormone conjugates, thyroid hormones, prostanoids, oligopeptides, and the drugs benzylpenicillin and pravastatin (Abe et al.
1999; Hsiang et al. 1999; Konig et al. 2000a). OATP8 was also cloned from liver and its protein was localized to the basolateral domain of human hepatocytes.
Functionally OATP8 transports BSP, steroid hormone conjugates, thyroid hormones (Konig et al. 2000a; Konig et al. 2000b). The OATP-D and OATP-E were cloned from human kidney and showed no homology with other members of OATP family.
The OATP-D transports estrone-3-sulfate, prostaglandin E2 and benzylpenicillin, and OATP-E transports in addition estradiol-17ß-glucuronide (Tamai et al. 2000). Only OATP-A has been tested for direct cortisol transport, which did not show any transport, while other members of OATP family members have not been tested for cortisol translocation (Bossuyt et al. 1996b; van Montfoort et al. 2003).
1.4.3 P-glycoprotein (Pgp) family
Another group of broad substrate specificity transporters is the P-glycoprotein family (Permeability-glycoprotein). P-glycoprotein (Pgp) plays an important role in multidrug resistance (MDR). The multidrug resistantce P-glycoprotein belongs to the subfamily B of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily of transporter proteins. Extensive studies have identified three classes of mammalian Pgps. Only two classes, class I and III, convey the MDR phenotype. Of the two
human genes, primarily the MDR1 (Chin et al. 1989) confers drug resistance (Ueda et al. 1987). P-glycoprotein acts as an energy-dependent efflux pump that exports anticancer agents out of the cell, lowering their intracellular concentration to sub-lethal levels, and is considered to be important in multidrug resistance of human tumors (Gottesman et al. 1988). P-glycoprotein is expressed in normal human tissues and is found on the luminal surface of transporting epithelia of the kidney proximal tubule, small intestine, colon, and liver biliary hepatocytes and in capillary endothelial cells of the brain and testis as well as in the adrenal cortex (Thiebaut et al. 1987; Thiebaut et al. 1989). The location of P-glycoprotein expression suggests that one of the physiological roles of P-glycoprotein is the secretion of metabolites and natural toxic substances into bile and urine and directly into the lumen of the gastrointestinal tract. It is important to identify the physiological substrates to predict the side effects that may arise from preventing the function of P-glycoprotein in chemotherapy, but no physiological substrates for P-glycoprotein to transport have been identified. Many reports on the cortisol interaction with MDR1 have been published (Farrell et al. 2000; Farrell et al. 2002; Karssen et al. 2001). Kalken and co-worker showed that the steroid hormones cortisol, testosterone, and progestrone cause an immediate, dose dependent increase of daunorubicin accumulation in Pgp overexpressing cells (Vankalken et al. 1993). These results showed the importance of MDR1 as a possible candidate of cortisol release from adrenal cells.